Proper functioning of the cell cycle and its regulation in response to both internal and external stimuli is essential for the normal growth, division, and differentiation of cells and tissues. Until recently biochemical study of these processes was difficult. The genetic and biochemical identification of the cyclin/p34cdc2 protein kinase complex as the key inducer of mitosis has opened this area to analysis. The activity of p34cdc2 is regulated by the binding of a regulatory protein, cyclin, and by both inhibitory and activating phosphorylations. These complex reactions are becoming increasingly amenable to study using purified components, an approach that should lead to deeper mechanistic insights into the cell cycle. The overall objective of the work proposed here is to understand this key transition in the cell cycle. The focus will be on how the activity of p34cdc2 is regulated by feedback and cell cycle inputs to control entry into mitosis. The long term approach will be to trace the regulatory pathways from direct effects on p34cdc2, through the enzymes that maintain the p34cdc2 phosphorylation state and their regulation, to the initiating signal. The Specific Aims of this project are: 1) To understand how the phosphorylation of p34cdc2 and the interactions of p34cdc2 with cyclin and with p13suc1 regulate its kinase activity. The prediction that phosphorylation within the putative ATP-binding site of p34cdc2 and the absence of cyclin prevent p34cdc2 from binding ATP will be tested by crosslinking studies. The existence of cell-cycle regulated protein binding to p34cdc2 will be explored and the role of one p34cdc2- binding protein, p13suc1, in controlling p34cdc2 phosphorylation state will be determined. 2) To study the specificity and regulation of the cdc25 phosphatase, and to clone the Xenopus homolog. cdc25 specifically dephosphorylates a tyrosine in p34cdc2. This dephosphorylation is required for p34cdc2 activation. 3) To identify, purify, and study the p34cdc2 activating kinase, the enzyme responsible for phosphorylating Thr-161 of p34cdc2, a site required for its activity. 4) To determine which of the six phosphorylation/dephosphorylation events on p34cdc2 are regulated during the cell cycle and between different cell cycle states. Regulated enzymes will be identified and studied to determine the biochemical basis for this cycle control.